Nature Protocols最新文献

{"title":"Synthesis of ordered mesoporous metal oxides by solvent evaporation-induced cooperative assembly.","authors":"Wenhe Xie, Xin-Yu Huang, Chengcheng Zhu, Jichun Li, Yu Deng, Youwen Rong, Keyu Chen, Yonghui Deng","doi":"10.1038/s41596-025-01225-w","DOIUrl":"https://doi.org/10.1038/s41596-025-01225-w","url":null,"abstract":"<p><p>Ordered mesoporous metal oxides (OMMOs) with periodically interconnected mesopores and crystalline framework have attracted ever-growing attention due to their high specific surface area, well-defined mesoscopic structures and adjustable pore-wall chemical microenvironment. It has been difficult to rationally design OMMO syntheses because the hydrolysis of metal salts is difficult to control; it is also difficult to find precursors that have a strong enough interaction with the structure-directing agents and oxides to overcome the formation of disordered metal oxide crystals rather than frameworks at the temperatures required for calcination. Here we describe an evaporation-induced cooperative assembly (EICA) approach for the controllable synthesis of high-quality OMMOs (for example, WO₃). The EICA approach endows precise control over the intermolecular interactions between metal oxide precursors and amphiphilic block copolymers such as poly(ethylene oxide)-block-polystyrene through ligand-assisted or cluster-involved assembly strategies and optimizes the thermal treatment process through carbon-supported crystallization. Based on this Protocol, a library of OMMOs with different framework compositions and desired pore sizes (10-35 nm, by changing the polystyrene length of poly(ethylene oxide)-block-polystyrene template) can be readily tuned, which can be further precisely modified by pore-wall engineering (for example, element doping, noble metal decoration and heterojunction construction). We describe the detailed experimental design and synthesis procedures to ensure the reproducibility of the experiments. Chemiresistive gas sensing and electrocatalytic hydrogen evolution reaction are introduced as potential applications of OMMOs. Except for the time (~2.5 d) needed for the preparation of amphiphilic block copolymers, the EICA approach for synthesizing OMMOs requires ~3.5 d without requiring special expertise.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144961973","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In situ structure determination of conformationally flexible targets using nextPYP.","authors":"Hsuan-Fu Liu, Ye Zhou, Qinwen Huang, Jeffrey Martin, Alberto Bartesaghi","doi":"10.1038/s41596-025-01218-9","DOIUrl":"10.1038/s41596-025-01218-9","url":null,"abstract":"<p><p>Single-particle cryoelectron tomography (SP-CET) is an imaging technique capable of determining the structure of proteins in their cellular environment at high-resolution. nextPYP is a web-based application designed to streamline the SP-CET structure determination process and facilitate the analysis of conformational variability. Here we explain how to use nextPYP-based methods to determine the structure and study the conformational heterogeneity of proteins using SP-CET. We provide a step-by-step guide to convert raw tilt-series into three-dimensional structures, following a workflow that includes movie-frame alignment, tilt-series alignment, contrast transfer function estimation, tomogram reconstruction, particle picking, high-resolution refinement and three-dimensional classification. The advantages of nextPYP are its ease-of-use and effectiveness at extracting high-resolution information, which, combined with its storage and compute efficiency, shortens time-to-structure from months to days. The complete procedure, including interactive data analysis and visualization, is fully integrated within the application, with no need for external software. Starting from raw tilt-series data, users will be able to determine a near-atomic resolution structure of human immunodeficiency virus 1 Gag protein, eight translational states of Escherichia coli 70S ribosomes and a structure of human 80S ribosomes from plasma-focused ion beam milled HeLa cells. This Protocol is intended as a resource for those who are new to SP-CET as well as more experienced users that want to streamline the process of in situ structure determination and heterogeneity analysis using nextPYP. The procedure requires 18 h to complete.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12370279/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144883220","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A universal protocol for ultrafast direct regeneration and upcycling of spent lithium-ion battery cathode materials.","authors":"Haocheng Ji, Junxiong Wang, Xiao Qiu, Hengyu Ren, Haoyu Xue, Hao Zhang, Guanjun Ji, Hui-Ming Cheng, Guangmin Zhou","doi":"10.1038/s41596-025-01234-9","DOIUrl":"10.1038/s41596-025-01234-9","url":null,"abstract":"<p><p>The rapid acceleration of global electrification has increased demand for sustainable energy storage, making lithium-ion batteries (LIBs) essential for various applications. However, their limited lifespan presents challenges related to resource waste and environmental risks. Unlike traditional metallurgical methods, which extract key metals from spent cathodes, the direct recycling process repairs damaged materials, maximizing their residual value through effective treatments. Despite widespread interest, systematic protocols to guide interdisciplinary researchers in direct recycling studies remain scarce. Using spent LiMn₂O₄ as an example, this protocol outlines a general approach for direct recycling and upcycling of spent LIBs. Initially, the failure condition of the spent cathode is evaluated using X-ray diffraction and inductively coupled plasma analysis to determine appropriate recycling parameters. The resulting recycled products include regenerated LiMn₂O₄ and upcycled next-generation cathode materials, such as high-voltage LiNi_0.5Mn_1.5O₄ and Co-free, Li-rich Li_1.2Ni_0.2Mn_0.6O₂. Subsequently, electron microscopy, spectroscopic techniques and electrochemical performance tests evaluate recycling effectiveness. This protocol incorporates two representative recycling methods to provide readers with a detailed procedural guide. Solid-phase regeneration forms the basis of most direct recycling technologies; thus, it requires minimal adjustments for broad applicability. Joule heating, a more emerging recycling technology, leverages rapid nonequilibrium reactions, substantially reducing processing time and introducing beneficial structural defects and elemental gradient distributions within the material. Compared to metallurgical methods, solid-phase and Joule heating-based protocols reduce recycling time to ~32 h and 5 h, respectively. Overall, this protocol provides a reliable guide for researchers, promoting sustainable LIB recycling and advancing clean energy research.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144874131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Systematic pegRNA design with PRIDICT2.0 and ePRIDICT for efficient prime editing.","authors":"Nicolas Mathis, Kim Fabiano Marquart, Ahmed Allam, Michael Krauthammer, Gerald Schwank","doi":"10.1038/s41596-025-01244-7","DOIUrl":"https://doi.org/10.1038/s41596-025-01244-7","url":null,"abstract":"<p><p>Prime editing is a versatile genome editing technology that enables precise genetic modifications without inducing DNA double-strand breaks. Owing to numerous variables in the prime editing guide RNA (pegRNA) design, experimentally identifying the most efficient pegRNA for a specific locus and edit is laborious. Therefore, we have developed computational tools to streamline this process. Here we present a comprehensive protocol detailing how to use PRIDICT2.0 and ePRIDICT, machine-learning models that assess the influence of the pegRNA design and chromatin context on prime editing. PRIDICT2.0 is an ensemble of attention-based bidirectional recurrent neural networks that predicts pegRNA efficiencies for replacements, insertions or deletions in different cellular contexts. Compared with other pegRNA design tools, PRIDICT2.0 accommodates larger edits-up to 40 base pairs-across diverse edit types, also allowing the introduction of silent bystander edits that can enhance editing efficiency. ePRIDICT, a gradient-boosting algorithm, further accounts for the local chromatin environments and assesses how the genomic location of the target site affects prime editing rates. Both tools are available at www.pridict.it for individual predictions or can be installed locally for batch processing of multiple edits and target sites. The protocol provides step-by-step instructions on using PRIDICT2.0 and ePRIDICT, covering sequence input, prediction generation and interpretation. Web-based predictions take under a minute, while local installation and batch processing may take up to several hours, depending on the dataset size. By streamlining pegRNA selection and chromatin context analysis, these tools promote the adoption of prime editing in basic and translational research.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855847","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Derivation, expansion and cryopreservation of primary fetal organoids from second and third trimester human amniotic fluid cells.","authors":"Giuseppe Calà, Giorgia D'Ariano, Kylin Yunyan Sun, Gloria Ji Zhang, Giuseppe Matteo Carrino, Alessandro Mariani, Carlotta Camilli, Isabella Fabietti, Roberto Bei, Anna L David, Alessandro Filippo Pellegata, Panicos Shangaris, Marco Pellegrini, Giovanni Giuseppe Giobbe, Paolo De Coppi, Mattia Francesco Maria Gerli","doi":"10.1038/s41596-025-01227-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01227-8","url":null,"abstract":"<p><p>Human primary fetal stem cell-derived organoids are used to model developing tissues in vitro. However, ethical and legislative constraints restrict fresh fetal tissue collection in several countries. Amniotic fluid (AF) is easily accessible with minimal ethical and regulatory constraints for collection. Our team recently showed that tissue-specific stem/progenitor cells can be isolated from fetal fluids collected during pregnancy through clinically indicated minimally invasive procedures conducted during the second and third trimesters. These samples consistently generate fetal lung, kidney tubule and gastrointestinal epithelial organoids autologous to the developing fetus. AF-derived organoids (AFOs) allow the investigation of fetal epithelia at developmentally relevant stages. Moreover, AFOs allow research to be conducted on late gestational stages, hardly accessible with other methods. Here, we provide a detailed protocol to establish, characterize and cryopreserve AFOs from viable AF cells. This includes the processing of patient-derived AF samples, viable cell sorting, seeding, establishment of clonal AFO lines, tissue phenotyping, expansion and cryopreservation. Additionally, we describe a straightforward immunofluorescence-based approach to pinpoint the tissue identity of the AFOs in a quick and cost-effective manner. In our hands, the protocol enabled the generation of primary fetal AFOs from 85.71% of samples (62.5% ascribed to the fetal lung, 59.4% to the kidney tubule and 6.2% to the small intestine). It takes 4-6 weeks to implement, requiring only standard equipment and expertise commonly available in cell biology laboratories.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144855846","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Modeling lymph node metastases in vivo.","authors":"Rodrigo J Gonzalez, Changwei Peng, Ulrich H von Andrian","doi":"10.1038/s41596-025-01235-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01235-8","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144847662","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spontaneous and experimental models of lymph node metastasis.","authors":"Cort B Breuer, Zhewen Xiong, Alice Wang, Grayson E Rodriguez, Gita C Abhiraman, K Christopher Garcia, Nathan E Reticker-Flynn","doi":"10.1038/s41596-025-01200-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01200-5","url":null,"abstract":"<p><p>Lymph node (LN) metastasis is a conserved feature across most solid organ malignancies and portends worse prognoses. Functionally, LN metastases induce systemic tumor-specific immune tolerance and may serve as a reservoir for distant metastases. Nonetheless, there are relatively few preclinical models for interrogating the biology of LN metastasis and its systemic effects at various stages of metastatic progression. We describe a method for modeling LN metastasis of melanoma tumors in mice that enables assessment of tumor and immune cell phenotypes and the functional roles of nodal involvement on distant metastasis. Our model comprises a family of transplantable syngeneic melanoma tumor cell lines evolved to exhibit enhanced LN metastatic potential, which can be used to probe cancer-immune interactions and test new therapeutics. We present both (i) a spontaneous LN metastasis model involving primary tumor implantation and assessment of LN colonization 21-28 d later and (ii) an experimental metastasis model involving implantation of primary tumors followed by direct intra-LN injections of tumor cells. Both models can be extended to assess the impact of LN metastasis on the development of distant metastases through asynchronous intravenous injections of tumors. Finally, we discuss experimental design considerations including when to use spontaneous or experimental models and troubleshooting consistent LN metastasis, making this model accessible for researchers with basic mouse survival-surgery skills. We highlight how LN metastasis models can be used to profile metastatic immune reprogramming, measure the impact of nodal metastases on distant metastases and assess novel anti-metastatic therapeutics.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144847663","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Targeted isolation of H₂-dependent methylotrophic methanogens by a cocktail approach.","authors":"Kejia Wu, Lei Zhou, Laiyan Liu, Min Yang, Jiang Li, Shichun Ma, Diana Z Sousa, Lei Cheng","doi":"10.1038/s41596-025-01224-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01224-x","url":null,"abstract":"<p><p>Methanogenic archaea play a crucial role in the global carbon cycle and in climate change. Recent metagenomic sequencing has revealed a considerable number of (putative) H₂-dependent methylotrophic methanogens (HMMs) across the archaeal tree and in diverse environments. Traditional isolation methods, such as dilution-to-extinction and roll-tube techniques, fail to cultivate fastidious HMMs. Here, we describe a four-stage isolation strategy designed to selectively isolate HMMs by using a flexible combination of methods to systematically reduce microbial complexity to a pure culture. In the initial stage, the growth conditions for the target HMM were optimized through closed-batch cultivation encompassing >50 conditions. Second, HMM-containing cultures were serially diluted in 96-well plates combined with substrate limitation to eliminate non-target archaea. In stage 3, the bacterial diversity in the culture was further decreased to a single bacterium by treatment with antibiotics and lysozyme. Finally, a last bacterial contaminant was removed by repeated addition of antibiotic mixtures and successive dilution transfers, leading to the successful isolation of the first pure culture of Methanosuratincola petrocarbonis LWZ-6, an HMM of the phylum Thermoproteota. This protocol also describes molecular methods, including 16S rRNA gene amplicon sequencing, metagenome sequencing and quantitative PCR, to track microbial community shifts and assess the growth advantage of the target HMM, enabling monitoring of the stepwise elimination of non-target microorganisms and ultimately confirming the purification of the target HMM. The duration of the protocol will vary for different HMMs depending on their substrate utilization, growth rate and method selection.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144835785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemogenetic detection and quantitation of H₂O₂ in living cells.","authors":"Mohammad Eid, Uladzimir Barayeu, Tobias P Dick","doi":"10.1038/s41596-025-01226-9","DOIUrl":"https://doi.org/10.1038/s41596-025-01226-9","url":null,"abstract":"<p><p>Hydrogen peroxide (H₂O₂) is a natural product of aerobic metabolism. It acts as a signaling molecule and regulates fundamental cellular functions. However, it has remained difficult to measure intracellular H₂O₂ with high specificity and in a quantitative manner. Here, we present a detailed protocol for a chemogenetic method that enables the detection and quantitation of H₂O₂ in living cells by converting intracellular H₂O₂ into fluorescent or luminescent signals. This is achieved by expressing the engineered heme peroxidase APEX2 in cells and subcellular locations of interest and by providing an appropriate fluorogenic or luminogenic substrate from outside. This method differs fundamentally from previously developed genetically encoded H₂O₂ probes; those are reversible and measure the balance between probe thiol oxidation and reduction. By contrast, APEX2 turns over its substrate irreversibly and therefore directly measures endogenous H₂O₂ availability. Our detailed step-by-step protocol covers the generation of APEX2-expressing cell lines, the implementation of fluorescent and luminescent measurements and examples for application. Ectopic expression of APEX2 can be achieved in 3 days, while the actual measurements typically require 1-2 h. This protocol is intended for entry-level scientists.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144822088","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanoscale spatiotemporal cluster analysis of expressed and endogenous proteins.","authors":"Rachel S Gormal, Tristan P Wallis, Alex J McCann, Kye Kudo, Anmin Jiang, Parnayan Syed, Shanley F Longfield, Rumelo Amor, Frédéric A Meunier","doi":"10.1038/s41596-025-01209-w","DOIUrl":"10.1038/s41596-025-01209-w","url":null,"abstract":"<p><p>Super-resolution microscopy has revolutionized the ability to investigate biological structures and processes, which are now accessible at nanoscale resolution. Recent advances in single-particle tracking (SPT) approaches have enabled researchers to study the intermolecular dynamics of individual proteins within their native environments in live cells. Fluorescent intrabody localization microscopy expands on existing SPT approaches such as SPT photoactivated localization microscopy by granting access to the nanoclustering dynamics of intracellular endogenous proteins through the use of single-domain nanobodies that can also differentiate between the conformational states of proteins. Here we detail how to perform single-molecule imaging of expressed proteins and nanobodies raised against endogenous proteins. We provide a streamlined analytical pipeline utilizing newly established clustering algorithms for extracting meaningful biological information. Nanoclustering analysis using spatiotemporal indexing is an open-source program with a user interface that enables the extraction of a range of dynamic nanoclustering metrics, including spatial and temporal information, from SPT data. This Protocol combines these single-molecule tracking and spatiotemporal clustering approaches into a comprehensive guide for researchers to achieve the precise localization of expressed and endogenous proteins and the characterization of their conformation-specific clustering behavior within subcellular compartments at nanoscale resolution. The procedure requires 2-4 d and is suitable for users with some prior experience in super-resolution microscopy and microscopy data analysis.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144804451","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}